The goal of this project in renewal remains to determine the nature of the membrane protein(s) responsible for the furosemide- sensitive Na-K-Cl cotransport system (fs system). This membrane transport system is widely found in animal cells and is a central component in the volume regulation of erythrocytes and other cells. It symmetrically mediates an electroneutral coupled flux of Na, K, and Cl in response to their summated electrochemical potential. Inhibition of this transport system is the mechanism by which furosemide and other potent diuretics act on renal tubule cells to induce salt and water loss by the kidney. The molecular basis and mechanism underlying the cotransport of Na, K, and Cl ions across any cell membrane is unknown. In this grant we propose to use a mutant cell line, a number of cell lines transformed with the mutant DNA, and a cosmid genomic DNA clone rescued from one transformant, all of which carry a mutation that now has been characterized extensively and shown to produce a dominant, qualitative, likely structural, alteration in the fs system; to purify and characterize further the protein(s) that make up this system; and to establish a mechanism by which the system might function. The nature of the mutant protein will be determined by analyzing the sequence of the mRNA encoding it. This mRNA will be isolated using the gene, which has already been cloned in a cosmid vector, as probe. The isolated mRNA sequence serves to yield the protein's primary amino acid sequence, directly, and, through analysis, suggests its secondary and tertiary structure as well. More important, it provides a means by which specific alterations can be produced in the cotransporter's structure for later analysis of their impact on function. A functional analysis can be achieved by expressing the in vitro-mutagenized message in either cultured cells or oocytes, and assaying cotransporter activity. In this manner a map of key functional regions on the protein can be defined: a functional anatomy of the carrier which must ultimately be melded into the kinetic model of its behavior. This project is a necessary step in the isolation and characterization of the protein(s) responsible for the cotransport mechanism, an important transport mechanism which has thus far eluded structural definition.